Conventionally known immuno assay using an antigen-antibody reaction include a one step or a two step noncompetitive sandwich method, and a competitive method.
Now, description will be given of an assay embodied in the one step non-competitive sandwich method for assaying the amount of an antigen contained in a sample, such as blood collected from a patient as a test sample. A sample to be assayed is added into a reaction cuvette. The reaction cuvette is previously charged with an antibody (hereinafter referred to as a "solid phase antibody") bound to an insoluble carrier (solid phase) such as the inside surface of a plastics cuvette or plastics particles, and an antibody (hereinafter referred to as "labeled antibody") bound to a labeling substance, such as a radioactive substance, a fluorescent substance, or an enzyme. In the reaction cuvette, an antigen contained in the sample reacts with the solid phase antibody and, as a result of this antigen-antibody reaction (immuno-reaction), an antigen-antibody complex is formed. At the same time, the labeled antibody is combined with the antigen-antibody complex, thus forming a complex composed of three components sandwiched together, namely, the solid phase antibody, antigen and labeled antibody.
Thus, the labeling substance of the labeled antibody is bound to the solid phase with the agency of the antigen in the sample.
Then, a process is performed to separate an excessive labeled antibody which is other than the labeling substance bound to the solid phase and which has not been bound to the antigen added into the reaction cuvette, and antibody components which have not taken part in the immuno-reaction (this operation being hereinafter referred to as "B/F separation"). Finally, the amount of the labeling substance proportional to the amount of the antigen bound to the solid phase is quantitatively assayed by a physical or a chemical technique by making use of properties of the labeling substance, thereby to determine the antigen concentration in the sample.
On the other hand, the two-step non-competitive sandwich method is an assay of the class wherein to a reaction cuvette previously charged with a solid phase antibody alone, a sample is added to perform a first reaction, and after that a labeled antibody is added into the reaction cuvette so as to perform a second reaction.
In other words, the sample is added into the reaction cuvette which is previously charged with the solid phase antibody (or a reagent containing therein the solid phase antibody). As a consequence, a particular antigen in the sample is bound and fixed to an insoluble carrier via an immuno-reaction with the solid phase antibody. Unreacted components that have caused no immuno-reaction are removed from the reaction cuvette through the B/F separation. Then, the labeled antibody is added into the reaction cuvette to cause an immuno-reaction through which a complex consisting of the solid phase antibody, the antigen, and the labeled antibody is formed. Unreacted components and residue are removed from the reaction cuvette via the B/F separation.
The foregoing operation is followed by an assay in which the amount of the complex bound to the insoluble carrier is assayed by quantitative determination of the amount of the labeling substance in the same manner as the aforesaid one-step method, so as to determine the antigen concentration of the sample.
Apart from the noncompetitive sandwich methods, the so-called competitive method is also known in which an antigen (generally referred to as "labeled antigen") which is labeled in advance with a labeling substance, and an antigen in a sample are competitively reacted with the aforesaid solid phase antibody.
According to the competitive method, the sample is put into reaction with the solid phase antibody and a reagent containing the labeled antigen. A component in the sample to be assayed (i.e., an antigen) and the labeled antigen take part in a competitive reaction with an immuno reactive portion of the solid phase antibody. As a result of this reaction, an unknown amount of antigen component contained in the sample and a predetermined amount of labeled antigen component, respectively, form immune complexes according to their ratio of amount (or ratio of concentration). Then, unreacted components and residue are removed by the B/F separation in the same manner as the sandwich method, and after that the amount of the labeling substance bound to the solid phase is assayed by quantitative determination using the properties of the labeling substance with the result that the amount of the antigen in the sample can be determined by calculation made in accordance with the ratio described above.
As previously described, assays of the amount of the labeling substance bound and fixed to the solid phase are differentiated by the properties of the labeling substance (may be also called "marker"). According to the classification prepared on the basis of this point of view, the immunoassay is frequently called, in an abbreviated form, "FIA" when involving the use of a fluorescent substance, "RIA" when involving the use of a radioactive substance, "EIA" when involving the use of an enzyme substance and "CLIA" when involving the use of a chemiluminescent substance as a labeling substance, respectively.
In the immunoassay of the type concerned a means is used for forming an immobilized solid phase. This means may include an antibody- or antigen-insolubilized carrier consisting of an antibody or an antigen fixed to tiny balls of synthetic resin such as polystyrene or polyvinyl chloride, tiny iron balls, tiny glass balls or tiny glass flakes (such a carrier is generally called "beads" or "particles", but in the description given below, the term "particles" is used to refer to the carrier).
In the aforesaid assays, an operation for supplying/discarding particles in the reaction cuvette, an operation for performing a washing for B/F separation, and an operation for replacing the particles should be performed at a predetermined timing. This requirement is too strict for the manual operations, and so various proposals for automated assays have been made (Japanese Patent Laid-open Publication No. 62-133355, Japanese Patent Laid-open Publication No. 62-133356, Japanese Patent Laid-open Publication No. 63-24160, Japanese Patent Laid-open Publication No. 63-24161, and Japanese Patent Laid-open Publication No. 3-51762). These prior proposals are, however, not fully satisfactory in terms of the way of timing.
The present invention was conceived in view of the foregoing prior art and has for its object the provision of an automatic immunoassay and an apparatus for the same which are capable of automatically performing a series of operations necessary for supplying and discarding particles in a reaction cuvette, for performing a washing for the B/F separation, for dispensing a reagent, and for performing an immunoassay, while allowing the reaction time to be changed freely, and which are readily practiced and embodied in the one step or the two step noncompetitive sandwich method as well as the competitive method.